Gene delivery is a promising therapeutic approach for a variety of diseases. However, the exact physical mechanisms of transfection agent mediated gene delivery are yet to be fully understood. The endosomal membrane is a major barrier for efficient transfection and endosome escape has become known as a crucial step in the delivery of nucleic acids. Previous research revealed distinct reagent-mediated membrane disruption mechanisms: the formation of small pores allowing protons to pass biological membranes and the permeabilization of large molecules such as LDH through amphiphilic translocation. Here, we measure the membrane permeation of protons in cultured cells after exposition to commercial transfection agents at endosomal pH conditions (pH 5.5) using a proton-sensing transistor (ISFET). In addition, we characterize the effect of transfection agents on cytosolic LDH leakage from cultured cells. Comparing the results from both assays at endosomal pH indicate that both types of transfection reagents have pore-forming activity at endosomal pH, while there is no such activity at pH 7.4. The pores formed by polymer-based reagents result in LDH leakage, whereas lipid-based reagents do not. This suggests a mechanistical difference in terms of the size of the pores formed. The effect of this difference on the endosomal escape profile is also investigated with a CLSM-based assay. These data indicate that the ISFET may be used to more accurately assess the endosome escape capabilities of different gene carriers.